During the reporting year ending September 30, 1998, essentially no experimental data were determined for the alpha-1-proteinase inhibitor (A-1-PI) folding/unfolding study, and the thermal stability of human albumin was not studied also. However, a manuscript was prepared on the basis of experimental work performed from August 1995 through September 1997 on the A-1-PI project, and the summary follows. A-1-PI regulates the activity of human neutrophil elastase. Functionally active A-1-PI (intact form) contains a mobile reactive site loop, which is an extension of strand 5 of the A beta-sheet. A-1-PI can also exist in other forms in which the loop is inserted between central strands of the A beta-sheet of the same molecule (latent form) or, more commonly, between those of another molecule to form loop-sheet polymers. The inserted forms show no inhibitory activity but are more stable than the intact form. However, folding in vivo favors formation of metastable, intact A-1-PI. Our results show that guanidine-HCl (Gu) induced unfolding of A-1-PI is a biphasic process with an intermediate state at 1.5 M Gu that can consist of one or more folding intermediates. Size exclusion-HPLC in 1.5 M Gu demonstrates that an equilibrium distribution of monomeric and polymeric intermediates slowly forms. The distribution is stable and the same in 1.5 M Gu whether starting with folded or fully unfolded inhibitor, and the relative concentration of polymeric intermediates increases with increase in A-1-PI concentration. Our circular dichroism data demonstrate that the intermediates have slightly relaxed secondary structures relative to those of the folded forms. Furthermore, the observed rate of folding from the intermediate state is slow and the same as that from the fully unfolded state thereby indicating that the folding of the intermediates is rate limiting and, thus, that this intermediate state is on the folding pathway. Even though intact A-1-PI is not the most stable form, it is the most kinetically accessible form during folding by dilution. As a result, folding A-1-PI by dilution maximizes the amount of active but less stable intact A-1-PI, whereas folding by step-wise equilibrium dialysis maximizes the amount of inactive but more stable polymer.